GSM7473984: hsSKM_ChIP_Normal Rabbit IgG _R1_rep2; Homo sapiens; ChIP-Seq
Growth medium was replaced with 6 ml of PBS and cells were cross-linked with 10 mM dithiobis(succinimidyl propionate) (DSP) in 6 ml PBS at room temperature for 30 min. Unreacted DSP was quenched by adding 0.18 ml of 1 M Tris-HCl pH=8.0 at room temperature for 15 min and the cells were rinsed with PBS once. Fixed cells were lysed with 400uL RIPA-G buffer + protease inhibitor cocktail (PIC) +RNase to extract DNA-protein complexes. The extract was centrifuged at 15,000 x g for 10 min at 4°C. The pellet was rinsed with 400 uL PBS 5 times to remove the lysis buffer and then suspended in 400 uL PBS by pippeting. After centrifugation at 10,000 x g for 2 min at 4°C, the pellet was redissolved in 100 uL RIPA-T buffer +RNase by sonication (1s/1s, 5s). The redissolved DNA-protein complex was digested with 0.5 uL Micrococcal Nuclease (#10011, Cell Signaling Technologies) at 37°C for 30 min. After centrifugation at 20,000 x g for 30 min at 4°C, the supernatant was collected in a new tube. In total, 600 uL of supernatant from six 10 cm dishes (100 uL each) was pooled. The supernatant was incubated with 2 ug rabbit anti-CBF-beta antibody (#62184s Cell Signaling Technologies)，5 ug mouse anti-YAP1 antibody (63.7) (sc-101199, Santa Cruz Biotechnologies) or 2 ug rabbit normal IgG (a technical negative control, #2729, Cell Signaling Technologies) overnight with rotation, then incubated with 30 uL Protein G Magnetic Beads (#9006) for 2 hours with rotation in accordance with SimpleChIP Enzymatic Chromatin IP kit (#9003; Cell Signaling Technologies)  with slight modifications as follows. The magnetic beads were pelleted by magnetic separation rack, and then washed 3 times with 1 mL low-salt buffer at 4°C for 5 min with rotation. The magnetic beads were washed once with 1 mL of high salt buffer at 4°C for 5 min with rotation and then incubated with 150 uL 1xChIP Elution buffer at 65°C with vortexing briefly every 2-3 min for 30 min in total. The magnetic beads mixture was incubated with 2 uL Proteinase K (#10012, Cell Signaling Technologies) and 10 mM DL-Dithiothreitol (PE001, LEAGENE) at 65°C for 2 hours to remove the bound proteins. The eluted DNA fragments were purified using E.Z.N.A. R Cycle-Pure Kit (Omega_D6492-02; Omega BIO-TEK) and subjected for library construction. The library construction used TruSeq DNA PCR-free prep kit (20015963, illumina). Library was sequenced by NovaSeq Sequencer.